Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is a foodborne diarrheal illness typically transmitted by contaminated beef or produce. EHEC produce Shiga toxin (Stx), a protein synthesis inhibitor that can cause severe disease, and upon systemic absorption, can lead to the devastating illness, hemolytic uremic syndrome (HUS). Because Stx is encoded by a lysogenic bacteriophage, antibiotic therapy is precluded, as antibiotics may induce the phage and increase toxin production, exacerbating disease. EHEC-mediated damage to the intestinal epithelium is critical for HUS by facilitating movement of Stx from the intestine into the bloodstream. Upon colonizing the intestinal epithelium, EHEC penetrates the mucus layer and injects effectors into host cells via a type 3 secretion system, triggering formation of ?attaching and effacing? (AE) lesions characterized by microvillar effacement and actin pedestal formation beneath bound bacteria. Stxs can induce proinflammatory responses in intoxicated cells and cause cell death. It has been postulated that EHEC disrupts intestinal barrier function when cytoskeletal rearrangements are induced by injected type 3- secreted effectors, by Stx-mediated enterocyte death, and/or by the influx of inflammatory cells. Unfortunately, we lack an understanding of how epithelial damage and subsequent toxin uptake occurs, in part because we lack models that mimic the complex disease-promoting interactions that occur in vivo between EHEC, Stx and the multiple host cell types. A recently developed 2-D colonoid model permits an evaluation of the contributions of bacteria, host cells, and the toxin itself in uptake of Stx into intestinal cells and transfer across the intestinal epithelium. Colonoid monolayers, derived from stem cells, polarize and differentiate into the four cell types that comprise human colon, so cell-specific virulence factor target(s) can be distinguished, and their apical and basolateral surfaces are accessible. Like in human colon, an apical adherent mucus layer is present. Inflammatory cells such as macrophages can be introduced, adding to the ability to study EHEC- colonic epithelial interactions in the presence of immune cells. The 2-D colonoid model allows for assessment of how individual virulence factors may work in concert to cause maximal intestinal damage. In this proposal, we identify intestinal epithelial targets of Stx and key events that promote toxin binding and uptake. We assess how Stx2 and outer membrane vesicles (OMV?s) containing Stx2 and other EHEC virulence factors interact with colonoid cells in the presence and absence of macrophages and EHEC. . We test whether intoxication by Stx itself influences these interactions.. Finally, we assess the effect of Stx, OMV?s, EHEC and/or macrophage co-culture on the apical junction complex, epithelial cell apoptosis, and proinflammatory molecule expression. By understanding the sequential proinflammatory, intestinal epithelium- damaging events that occur during EHEC infection that lead to systemic uptake of Stx, we hope to identify which of these events may be critical and drug-targetable, thus preventing HUS.